The present invention relates to the field of quantitative microspectroscopy, and in particular to a method for calibrating the height of a sample in a sample analyzer.
The determination of such blood parameters as the Hematocrit (xe2x80x9cHCTxe2x80x9d ), the Volume of single Red Blood Cells (xe2x80x9cRCVxe2x80x9d ), the Mean Cell Volume (xe2x80x9cMCVxe2x80x9d ) and the Red Cell Distribution Width (xe2x80x9cRDWxe2x80x9d ) are of eminent clinical interest. Usually, systems based on electrical impedance measurement (Coulter Counter) or based on light scattering (Flow Cytometer) are employed (see. e.g., J. B. Henry, xe2x80x9cClinical diagnosis and management by laboratory methodsxe2x80x9d, W. B. Saunders Company, Philadelphia, 1996, pp. 548 ff. or D. H. Tycko, M. H. Metz, E. A. Epstein, A. Grinbaum, xe2x80x9cFlow-cytometric light scattering measurement of red blood cell volume and hemoglobin concentrationxe2x80x9d, Applied Optics 24 (1985), 1355-1365). Impedance counters are complex and expensive instruments that require very careful adjustment and control of instrument and sample parameters. A major disadvantage of flow cytometers is the fact that the parameters of light scattering depend not only on cell volume, but also on the cell""s shape.
In 1983, Gray, Hoffman and Hansen proposed a new optical method for determining the volume of cells in a flow cytometer (M. L. Gray, R. A. Hoffman, W. P. Hansen, xe2x80x9cA new method for cell volume measurement based on volume exclusion of a fluorescent dyexe2x80x9d, Cytometry 3 (1983), 428-432). In this method, the cells are suspended in a fluorescent dye, which is unable to penetrate the cell membrane. The level of fluorescence which is produced when a narrow stream of the cell suspension is excited by a focused laser beam will remain constant until a cell arrives in the illuminated region thereby causing a decrease in fluorescence intensity which is directly proportional to the cell""s volume. In a flow cytometer, a single cell is passing through the laser-illuminated spot within approximately 10 xcexcs. Due to this short data acquisition time interval, the electronic detection bandwidth has to be relatively large, which results in a poor signal-to-noise ratio and in a low precision for the volume determination.
The available data acquisition time can be significantly increased by suspending the cells in a stationary sample and applying digital imaging fluorescence microscopy (see P. L. Becker, F. S. Fay, xe2x80x9cCell-volume measurement using the digital imaging fluorescence microscopexe2x80x9d, Biophysical Journal 49 (1986), A465). In the digital fluorescence microscopy approach, a calibration procedure is required in order to determine the cell volume. Recktenwald and co-workers have introduced a method where the calibration is performed by means of optical transparent and non-fluorescent microspheres that are suspended together with the cells (D. Recktenwald, J. Phi-Wilson, B. Verwer, xe2x80x9cFluorescence quantitation using digital microscopyxe2x80x9d, Journal Physical Chemistry 97 (1993), 2868-2870). The volume of individual spheres is determined by measuring their projection area under the microscope and transforming this number into a volume, assuming an ideal spherical shape. The decrease in fluorescence intensity as a result of the spheres"" volume that is being excluded from emitting fluorescence is used as the required calibration parameter. The advantage of this approach is given by the fact that the calibrating particles are located within the sample itself. In other words, a calibration is performed on the very same sample container, and no extra calibration sample is required.
The use of calibration spheres within a cell suspension is not without problems. First, the introduction of the spheres represents an additional step in the workflow. In systems that are designed for high throughput, this additional step would represent a disadvantage. Secondly, Recktenwald and co-workers observed a tendency of the fluorescent dye molecules to settle down on the sphere""s surface, which causes an error. Third, if the optical index of refraction of the spheres does not match well with the liquid""s index, then refraction-based artifacts in the measured fluorescence intensity occur at the edges of the spheres. And, finally, the use of microspheres can represent a problem, if e.g. a thin sample thickness in the order of a few micrometers or less is needed.
In order to overcome the problems in the prior art, it has been suggested (U.S. Pat. No. 6,127,184 to Wardlaw) to design a cuvette-like optical sample container for the cell suspension that has different optical pathlengths in different areas. In at least one area, the thickness of the liquid layer of undiluted blood is so thin (2 to 7 microns) that monolayers of isolated Red Blood Cells (xe2x80x9cRBCsxe2x80x9d ) are formed. In another region, the liquid layer is thicker (7 to 40 microns), and typical chain-like aggregates of RBCs (referred to as xe2x80x9cRoleauxxe2x80x9d are forming. The thick area is used to determine the HCT, and the thin area is used to determine the RCV. As in the prior art, the blood plasma is stained with a fluorescent dye that is not penetrating into the RBCs.
In a method and apparatus described in U.S. Pat. No. 6,127,184, the optical cuvette is placed under a microscope, illuminated with excitation light, and reemerging fluorescence is measured by means of an imaging photodetector such as a CCD camera. The RCV is determined using the equation   RCV  =            [              1        -                              B            RBC                                B            P                              ]        *    A    *    d  
where BP is the fluorescence intensity emerging from an area of known size, A, within a cell-free plasma region. BRBC is the fluorescence intensity emerging from another area of same size, but comprising a single RBC. In practice, BP is determined by measuring the fluorescence intensity in a cell-free region near a particular RBC, and by extrapolating to the full size, A. In contrast to the HCT determination, the absolute area, A, and the absolute height of the liquid layer, d, have to be known. One could also say that the absolute volume
V=A*d
in which the single RBC is embedded, has to be known. The area, A, can be easily determined under the microscope. The determination of the height, d, near the RBC is a more complicated problem and is named xe2x80x9ccalibrationxe2x80x9d .
U.S. Pat. No. 6,127,184 discloses some methods to xe2x80x9ccalibratexe2x80x9d the optical cuvette, i.e., to determine the height, d. In one method, a square-shaped capillary of known volume is integrated into the cuvette. By measuring the fluorescence intensity reemerging from this capillary, one could obtain a calibration parameter C=intensity/volume. Since the fluorescence intensity per unit area is assumed to be proportional to the height of the cuvette, the height at any location can then be determined via the reemerging fluorescence intensity. However, in practice, integrating a small part such as a pre-fabricated capillary into a plastic disposable can be difficult and costly.
Other methods disclosed in U.S. Pat. No. 6,127,184 include using a step-like change in the cuvette thickness as a means for calibration, and using a molded calibration standard such as a well of accurately controlled height. However, these methods have some drawbacks in that they require the additional step of precision molding which can be difficult and costly.
Consequently, it has been found that there exists a need for calibrating the height of a sample in a sample analyzer, that would not require molded calibration tools of extreme precision.
It is an objective of the present invention to provide a method for calibrating the sample height in a sample analyzer, and in particular a calibration method that would be exact, but would not require molded calibration tools of extreme precision within the sample analyzer.
According to the present invention, the above objective is achieved by depositing a sample of biological fluid, and preferably, whole blood into a chamber, such as for example, an optical cuvette, whereby the blood plasma contains a dye and preferably, a fluorescent dye, that does not diffuse into the red blood cells. The sample is illuminated with excitation light so that the plasma emits fluorescence radiation, which is detected by, e.g., a microscope""s imaging photodetector. The fluorescent dye is selected so that neither the excitation light nor the emitted fluorescence light are absorbed significantly by the red blood cells.
A height value in the single-cell area is then determined by performing the process steps of (a) measuring fluorescence intensity values in cell-free locations within the single-cell area as a function of the focal plane position, (b) determining the full-width-at-half-maximum (xe2x80x9cFWHMxe2x80x9d) of the fluorescence-intensity-versus-focal-plane-position curve, and (c) calculating the height, d, in the single cell area from the quantity FWHM by taking into account the index of refraction of the blood plasma.